Objectives and Health Related Issues. The intoxicating effects associated with exposure to ethanol have been linked to changes in the activities of several neurotransmitter receptors that are ligand-gated ion-channels. Acute exposure of the gamma-amino-butyric acid (GABA) and N-methyl D-aspartate (NMDA) receptors to ethanol is directly implicated in the development of fetal alcohol syndrome and also in alcohol toxicity and alcohol dependency in the adult. There is increasing evidence that ethanol binds to specific sites on these receptors and induces a conformational change that modifies their activity. Characterization of alcohol-binding sites in ethanol sensitive proteins would provide potential targets for the development of pharmacological agents to control alcohol intoxication and alcohol dependency. At present there is no direct structural information available about the nature of potential binding sites of these important ethanol-sensitive proteins because of the inherent difficulties in studying integral membrane proteins. LUSH is a novel alcohol-binding protein from fruit flies that recognizes ethanol, n-propanol and n-butanol. We have recently solved the structure of LUSH in the complex with ethanol. The long-term goal of this proposal is to characterize the molecular nature of this binding site in detail in order to define the molecular basis for alcohol-binding specificity. The structure of LUSH bound to a series of alcohols will be solved using X-ray crystallographic methods to reveal a molecular picture of alcohol specificity in a nonenzymatic protein. The effect of different alcohols on binding affinity and protein stability will be analyzed using biophysical and spectroscopic methods, and the differences correlated with changes to the protein structure in solution. The role of specific amino acids in alcohol binding and protein function will be tested using site directed mutagenesis to engineer proteins with modified ligand-binding properties. The ultimate goal is to develop a model for specific alcohol-binding sites in alcohol-sensitive proteins that will aid in an understanding of the molecular basis of alcohols actions in causing intoxication and toxicity.